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Mubarak-Ali, Al-Fahim, Rahiwan Nazar Romli, and Nilam Nur Amir Sjarif. "Code Clone Detection Model: A SWOT Analysis Perspective." Advanced Science Letters 24, no. 10 (October 1, 2018): 7210–13. http://dx.doi.org/10.1166/asl.2018.12916.
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Akhin, Marat, and Vladimir Itsykson. "Tree Slicing in Clone Detection: Syntactic Analysis Made (Semi)-Semantic." Modeling and Analysis of Information Systems 19, no. 6 (March 12, 2015): 69–78. http://dx.doi.org/10.18255/1818-1015-2012-6-69-78.
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Nowadays most of software contains code duplication that leads to serious problems in software maintenance. A lot of different clone detection approaches have been proposed over the years to deal with this problem, but almost all of them do not consider semantic properties of the source code. We propose to reinforce traditional tree-based clone detection algorithms by using additional information about variable slices. This allows to find intertwined/gapped clones on variables; preliminary evaluation confirms applicability of our approach to real-world software.
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Sotolongo, Ricardo, Fangyan Dong, and Kaoru Hirota. "Semantically Enhanced Code Clone Refinement Algorithm Based on Analysis of Multiple Detection Reports." Journal of Advanced Computational Intelligence and Intelligent Informatics 15, no. 3 (May 20, 2011): 322–28. http://dx.doi.org/10.20965/jaciii.2011.p0322.
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An algorithm based on semantic analysis of multiple detection tools’ reports using WordNet is proposed oriented on the refinement of code clones. It parses different detection tools’ reports looking for new clone specifications, and refines the location of existing ones using semantic information contained in source code. It is applied to a real and complex software system and is compared to three other well-known detection algorithms, discovering 4888 clone pairs more than the average detected by other tools; also making the code clones 3 lines longer (for a subset of the same system the results are proportional to the size reduction). The objective is to provide higher quantity of code clones, and more appropriated localization to be used in refactoring processes.
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Bartoszuk, Maciej, and Marek Gagolewski. "SimilaR: R Code Clone and Plagiarism Detection." R Journal 12, no. 1 (2020): 367. http://dx.doi.org/10.32614/rj-2020-017.
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Sargsyan, Sevak, Shamil Kurmnagaleev, Andrey Belevantsev, Hayk Aslanyan, and Artiom Baloian. "Scalable code clone detection tool based on semantic analysis." Proceedings of the Institute for System Programming of the RAS 27, no. 1 (2015): 39–50. http://dx.doi.org/10.15514/ispras-2015-27(1)-3.
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Lalar, Sacachin, Shashi Bhushan, and Surender Surender. "Analysis of Clone Detection Approaches in Static Wireless Sensor Networks." Oriental journal of computer science and technology 10, no. 3 (August 5, 2017): 653–59. http://dx.doi.org/10.13005/ojcst/10.03.14.
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Wireless Sensor Networks (WSNs) are developing very fast in the wireless networks. The wireless sensor network has the characteristics of limited memory, small size and limited battery. WSNs are vulnerable to the different types of attacks due to its characteristics. One of the attacks is clone node attack in which attacker capture the nodes from the network and stoles the information from it and replicates it in the network. From the clone nodes, the attacker can easily launch the different type of attacks in the network. To detect the clone node, different methods has been implemented .Each method having advantages and limitations. In the this paper, we explain the different methods to detect the clone nodes in the static wireless sensor network and compare their performance based on the communication cost and memory.
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Ma, Yu-Seung, and Duk-Kyun Woo. "Domain Analysis of Device Drivers Using Code Clone Detection Method." ETRI Journal 30, no. 3 (June 9, 2008): 394–402. http://dx.doi.org/10.4218/etrij.08.0107.0204.
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Haferlach, Claudia, Susanne Schnittger, Alice Fabarius, Armin Leitner, Wolfgang Kern, Ulrike Bacher, Rüdiger Hehlmann, Andreas Hochhaus, and Torsten Haferlach. "Analysis of Philadelphia Negative Clones Detected during Treatment with Tyrosine Kinase Inhibitors: A Study on 63 CML Cases." Blood 110, no. 11 (November 16, 2007): 4541. http://dx.doi.org/10.1182/blood.v110.11.4541.4541.
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Abstract The occurrence of Philadelphia chromosome negative (Ph−) clones in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors have been reported in approximately 5% of cases. The pathogenesis of this phenomenon still remains unclear. The clinical relevance of these new clones remains to be clarified, as only occasional reports describe the presence of hematological dysplastic features or development of overt disease such as MDS or AML. We found 63 patients with CML that developed Ph− clones and performed in total 281 chromosome analyses (median: 4 analyses per case; range, 1–18). In total, 66 clonal abnormalities were detected. 60 cases showed only one aberration, in the remaining 3 cases 2 abnormalities were detected. Remarkably, no complex aberrant karyotypes were observed. Most frequent aberrations were gains and losses of whole chromosomes: +8 (n=35, 55.6%), +Y (n=3, 4.8%), +11 (n=2, 3.2%), +X (n=1, 1.6%), −Y (n=9, 14.3%), −7 (n=6, 9.5%). The following abnormalities were only observed in a single case: inv(Y)(p11.1q11.2); +1,der(1;7)(q10;p10); del(5)(q13q33); der(7)del(7)(p13)del(7)(q11.2); del(7)(q11q22); der(7;15)(q10;q10); t(8;11)(q22;q23); del(12)(p11p13); del(20)(q11q13). The majority of aberrations were unbalanced, only 2 balanced rearrangements were observed. No clonal evolution was found. Although this pattern of abnormalities resembles closest the pattern observed in MDS or Ph− chronic myeloproliferative disorder, only 1 case with −7 developed a MDS and subsequently an AML. Most frequently in addition to the Ph− clone a Ph+ clone and a normal clone was observed (n=86). In 8 analyses the Ph− clone was the only clone detected and in 60 analyses the Ph− clone was accompanied by a normal clone, in 10 by a Ph+ clone. In one case two different Ph− clones were observed during the course of the disease. For 34 patients detailed clinical data are available. All these patients were treated with imatinib, 7 patients subsequently received dasatinib and 3 nilotinib after imatinib treatment. The Ph− clone was observed after a median of 43 months (mo) after diagnosis and 20.5 mo after start of imatinib treatment, respectively. Dasatinib treatment was started 2, 3, 6, 10 and 12 mo prior to the first detection of the Ph− clone and in 1 case 5 mo after occurrence of the Ph− clone. Nilotinib treatment was started 6, 7 and 11 mo prior to the first detection of the Ph− clone. In 15 cases imatinib treatment was started within the first 4 mo after diagnosis. In these cases the Ph− clone was observed in median 13 mo after start of imatinib treatment (range: 4–64). Overall the 34 cases were monitored for 428 mo after occurrence of the Ph− clone (median=11.5 mo). In 6 cases the Ph− clone was lost during follow-up (in one case after allogeneic SCT). In conclusion: Ph− clones are stable over time in most cases. In the majority of cases only single, usually unbalanced abnormalities are observed. The size of the Ph− clone fluctuates and can also disappear. In most cases the Ph− clones seem to have no clinical impact. Longer follow-up is necessary to clarifiy the prognostic impact. So far the available data do not imply that the occurrence of Ph− clones per se should lead to changes in treatment strategy. However, close cytogenetic monitoring is recommended.
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Kaur, Gundeep, and Sumit Sharma. "Metric level based code clone detection using optimized code manager." International Journal of Engineering & Technology 7, no. 2.27 (August 6, 2018): 144. http://dx.doi.org/10.14419/ijet.v7i2.27.13763.
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Object-oriented programming today, is the main prototype in typical software development. Code Cloning defines generally, all through the designing and development of software systems. Detection can be based on Textual analysis, Lexical analysis, Syntax analysis, Semantic analysis, Hybrid analysis and Metric analysis. The major drawback of the present research is that it focuses more on fragments of copied code and does not focus on the aspect that the fragments of duplicated code are may be part of a larger replicated program structure. In this process, techniques take a lot of time and it creates complexity. In our research, a source code is then scanned for detecting various methods by adopting a “OPTIMIZED SVM ALGORITHM” and the method definitions are extracted and collected by means of a CLONE CODE and saved for further reference. To evaluate the performance parameters we calculate the LOC, the number of repetitions, and maximum and minimum LOC. To enhance the performance metrics precision recall, accuracy and reduce the error rate and time complexity
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Aktas, Mehmet S., and Mustafa Kapdan. "Structural Code Clone Detection Methodology Using Software Metrics." International Journal of Software Engineering and Knowledge Engineering 26, no. 02 (March 2016): 307–32. http://dx.doi.org/10.1142/s0218194016500133.
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Unnecessary repeated codes, also known as code clones, have not been well documented and are difficult to maintain. Code clones may become an important problem in the software development cycle, since any detected error must be fixed in all occurrences. This condition significantly increases software maintenance costs and requires effort/duration for understanding the code. This research introduces a novel methodology to minimize or prevent the code cloning problem in software projects. In particular, this manuscript is focused on the detection of structural code clones, which are defined as similarity in software structure such as design patterns. Our proposed methodology provides a solution to the class-level structural code clone detection problem. We introduce a novel software architecture that provides unification of different software quality analysis tools that take measurements for software metrics for structural code clone detection. We present an empirical evaluation of our approach and investigate its practical usefulness. We conduct a user study using human judges to detect structural code clones in three different open-source software projects. We apply our methodology to the same projects and compare results. The results show that our proposed solution is able to show high consistency compared with the results reached by the human judges. The outcome of this study also indicates that a uniform structural code clone detection system can be built on top of different software quality tools, where each tool takes measurements of different object-oriented software metrics.
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Theja, G. Ravi, and C. Subhas. "Energy Consumption Analysis Of Clone Detection Approaches In Wireless Sensor Networks." i-manager’s Journal on Wireless Communication Networks 3, no. 2 (September 15, 2014): 10–16. http://dx.doi.org/10.26634/jwcn.3.2.2874.
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Ilyas, Muhammad, Hafiz Anas, Muhammad Hummayun, Mubeen Rafi, and Almas Kanwal. "A Comparative Analysis of Clone Detection Tools: Solid SDD and CCFinderX." International Journal of Computer Applications 148, no. 14 (August 22, 2016): 11–16. http://dx.doi.org/10.5120/ijca2016910912.
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Morley, Alexander, Michael Brisco, Sue Latham, Vicki Wilczek, Elisabeth Hughes, Brad Budgen, Rosemary Sutton, Anita Bahar, Maria Malek, and Glenn Marshall. "Markers for Minimal Residual Disease (MRD) in B-Lineage Acute Lymphoblastic Leukemia (B-ALL): Analysis of IgH Gene Rearrangement Repertoire (AIRR) vs Conventional Strategy." Blood 110, no. 11 (November 16, 2007): 4243. http://dx.doi.org/10.1182/blood.v110.11.4243.4243.
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Abstract Background: Detection of molecular markers for monitoring MRD in B-ALL conventionally involves searching for a variety of immunoglobulin and T-cell receptor rearrangements. However preliminary studies have suggested that AIRR may have advantages. Aim :To directly compare AIRR to the conventional approach for detection of molecular markers. Materials and Methods Marrow samples from a consecutive series of 50 children with B-linage ALL were studied by the conventional approach in Sydney and by AIRR in Adelaide. AIRR involves use of primers specific for individual V and J segments of the IgH gene followed by sequencing of amplified products and calculation of the relative abundance of rearrangements from the quantitative PCR data. IgH rearrangements are regarded as suitable for detecting the major leukemic clone if they comprise > 10% of the rearrangements present. Incomplete D-J rearrangements are detected using primers directed towards genomic regions upstream of the D segments. Results Table 1 shows the number of patients with either 0, 1 or 2 rearrangements suitable for monitoring MRD of the major leukemic clone. The 2 approaches appeared to be equivalent in terms of the number of markers for the major clone, but, of the rearrangements detected conventionally, 49% were IgH rearrangements and 51% involved IgK or various T-cell receptor genes. All of the IgH rearrangements detected in the 50 patients, grouped by abundance, are shown in Table 2. Table 1 number of patients no marker 1 marker 2 markers conventional approach 2 9 39 AIRR 2 12 36 Table 2 relative abundance of rearrangement 100-10% 10-1% <1% detected by both analyses 65 6 6 conventional only 4 0 0 AIRR only 28 12 31 High and low abundance rearrangements mark major and minor clones repectively. AIRR detected significantly more rearrangements of both types. Clone sizes and lineage relationships could be inferred from rearrangement abundance and sequence relationships. 68 minor clones were detected at diagnosis in 31 patients. 26 of the 68 showed no sequence relationship to the major clone. Discussion Use of IgH gene rearrangements as markers enables MRD in B-ALL to be sensitively quantified, down to 10−6. The superior ability of AIRR to identify marker IgH rearrangements is therefore likely to enable more sensitive monitoring of the major leukemic clone. Relapse in B-ALL is sometimes due a minor chemoresistant leukemic clone which is already present at diagnosis but is only identified retrospectively. The ability of AIRR to identify IgH markers for minor clones at diagnosis, together with the ability to sensitively monitor clones marked by IgH rearrangements, may enable prospective identification of these minor clones. Conclusions AIRR and the conventional strategy are similar in terms of the number of patients with ALL in whom 1 or 2 molecular markers for the major leukemic clone can be detected. AIRR detects significantly more IgH markers for the major leukemic clone. This should enable more sensitive quantification of the major clone in many patients. AIRR is much superior at identification of markers for minor clones. This may enable chemoresistant minor clones to be detected prospectively. The clonal hierarchies of minor clones at diagnosis resemble those observed in patients who relapse.
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Badve, Sunil, I. Tudor Vladislav, Betsy Spaulding, Anna Strickland, Sylvia Hernandez, Lisa Bird-Turner, Cecelia Dodson, Bjorn Elleby, and Therese Phillips. "EP1: a novel rabbit monoclonal antibody for detection of oestrogen receptor α." Journal of Clinical Pathology 66, no. 12 (July 26, 2013): 1051–57. http://dx.doi.org/10.1136/jclinpath-2012-201391.
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AimsAssessment of hormone receptor expression is part of routine examination of every breast cancer. In this study, we report the characterisation of a novel rabbit monoclonal antibody, clone EP1, directed against oestrogen receptor (ER) α. Additionally, its immunohistochemical performance characteristics in archival tissues are evaluated in normal tissues and two distinct cohorts of breast cancer patients.MethodsComparative analyses between EP1 and the anti-ERα component of the ER/PR pharmDx kit (cocktail of mouse monoclonal antibody clones 1D5 and ER-2-123) and between EP1 and another commercially available rabbit monoclonal antibody, clone SP1, are described.ResultsClone EP1 specifically detects nuclear ER in all tissues examined; cytoplasmic staining was not observed. The analysis shows a high degree of concordance (∼95%) between EP1 and both the ERα component of the Dako ER/PR pharmDx kit and Ventana clone SP1. However, the use of EP1 antibody together with Dako EnVision FLEX detection system resulted in a stronger staining intensity as compared with SP1 antibody using the Ventana ultraView DAB detection system resulting in better ‘ease of use.’ConclusionsThe use of EPI can result in better interpretation of the results of the ER analysis.
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Zhang, Zhihua, Shoushan Luo, Hongliang Zhu, and Yang Xin. "A Clone Detection Algorithm with Low Resource Expenditure for Wireless Sensor Networks." Journal of Sensors 2018 (2018): 1–16. http://dx.doi.org/10.1155/2018/4396381.
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Wireless sensor networks (WSNs) are facing the threats of clone attacks which can launch a variety of other attacks to control or damage the networks. In this paper, a novel distributed clone detection protocol with low resource expenditure is proposed for randomly deployed networks. The method consisting of witness chain establishment and clone detection route generation is implemented in the nonhotspot area of the network organized in a ring structure, which balances the resource consumption in the whole network. The witness chains and detection routes are in the centrifugal direction and circumferential direction, respectively, which can ensure the encounter of witnesses and detection routes of nodes with the same ID but different positions to detect clone attacks. Theoretical analysis demonstrates that the detection probability can be up to 1 with reliable witnesses. Moreover, both theoretical analysis and simulation results manifest that the proposed method can achieve better network lifetime and storage requirements with low resource expenditure and outperforms most methods in the literature.
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Bharti, Sarveshwar, and Hardeep Singh. "Metaphorical Analysis of Software Clone Detection Techniques based on Dimensions and Metrics." International Journal of Computer Sciences and Engineering 6, no. 12 (December 31, 2018): 151–56. http://dx.doi.org/10.26438/ijcse/v6i12.151156.
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Kaur, Harpreet, and Raman Maini. "Performance Evaluation and Comparative Analysis of Code-Clone-Detection Techniques and Tools." International Journal of Software Engineering and Its Applications 11, no. 3 (March 31, 2017): 31–50. http://dx.doi.org/10.14257/ijseia.2017.11.3.04.
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Polovikova, O. N., and V. E. Ivanova. "Clone detection in student programs based on lexical analysis of source codes." Journal of Physics: Conference Series 1615 (August 2020): 012021. http://dx.doi.org/10.1088/1742-6596/1615/1/012021.
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Cho, Kwantae, Minho Jo, Taekyoung Kwon, Hsiao-Hwa Chen, and Dong Hoon Lee. "Classification and Experimental Analysis for Clone Detection Approaches in Wireless Sensor Networks." IEEE Systems Journal 7, no. 1 (March 2013): 26–35. http://dx.doi.org/10.1109/jsyst.2012.2188689.
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Dulau-Florea, Alina E., Neal S. Young, Irina Maric, Katherine R. Calvo, Cynthia E. Dunbar, Danielle M. Townsley, Thomas Winkler, et al. "Bone Marrow as a Source of Cells for Paroxysmal Nocturnal Hemoglobinuria Detection." American Journal of Clinical Pathology 150, no. 3 (July 5, 2018): 273–82. http://dx.doi.org/10.1093/ajcp/aqy053.
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Abstract Objectives To determine fluorescently labeled aerolysin (FLAER) binding and glycophosphatidylinositol–anchored protein expression in bone marrow (BM) cells of healthy volunteers and patients with paroxysmal nocturnal hemoglobinuria (PNH) detected in peripheral blood (PB); compare PNH clone size in BM and PB; and detect PNH in BM by commonly used antibodies. Methods Flow cytometry analysis of FLAER binding to leukocytes and expression of CD55/CD59 in erythrocytes. Analysis of CD16 in neutrophils and CD14 in monocytes in BM. Results FLAER binds to all normal BM leukocytes, and binding increases with cell maturation. In PNH, lymphocytic clones are consistently smaller than clones of other BM cells. PNH clones are detectable in mature BM leukocytes with high specificity and sensitivity using common antibodies. Conclusions PNH clone sizes measured in mature BM leukocytes and in PB are comparable, making BM suitable for PNH assessment. We further demonstrate that commonly used reagents (not FLAER or CD55/CD59) can reliably identify abnormalities of BM neutrophils and monocytes consistent with PNH cells.
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Kapila, Ritu, Sandip Das, Malathi Lakshmikumaran, and P. S. Srivastava. "A novel species-specific tandem repeat DNA family from Sinapis arvensis: detection of telomere-like sequences." Genome 39, no. 4 (August 1, 1996): 758–66. http://dx.doi.org/10.1139/g96-095.
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DNA sequences representing a tandemly repeated DNA family of the Sinapis arvensis genome were cloned and characterized. The 700-bp tandem repeat family is represented by two clones, pSA35 and pSA52, which are 697 and 709 bp in length, respectively. Dot matrix analysis of the sequences indicates the presence of repeated elements within each monomeric unit. Sequence analysis of the repetitive region of clones pSA35 and pSA52 shows that there are several copies of a 7-bp repeat element organized in tandem. The consensus sequence of this repeat element is 5′-TTTAGGG-3′. These elements are highly mutated and the difference in length between the two clones is due to different copy numbers of these elements. The repetitive region of clone pSA35 has 26 copies of the element TTTAGGG, whereas clone pSA52 has 28 copies. The repetitive region in both clones is flanked on either side by inverted repeats that may be footprints of a transposition event. Sequence comparison indicates that the element TTTAGGG is identical to telomeric repeats present in Arabidopsis, maize, tomato, and other plants. However, Bal31digestion kinetics indicates non-telomeric localization of the 700-bp tandem repeats. The clones represent a novel repeat family as (i) they contain telomere-like motifs as subrepeats within each unit; and (ii) they do not hybridize to related crucifers and are species-specific in nature. Key words : Brassica species, Sinapis arvensis, tandem repeats, telomeres.
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AlGhasham, Nahlah, Yasmeen Abulkhair, and Salem Khalil. "Flow Cytometry Screening for Paroxysmal Nocturnal Hemoglobinuria Abnormal Clones in Saudi Arabia." Blood 124, no. 21 (December 6, 2014): 5162. http://dx.doi.org/10.1182/blood.v124.21.5162.5162.
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Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with insidious process, chronic course and life-threatening condition. PNH is clinically defined by the deficiency of the endogenous glycosyl phosphatidylinositol (GPI)-anchored complement inhibitory protein. It has always aroused interest in the medical profession rendering screening and proper diagnosis by flow cytometry (FCM) technology a priority We reviewed all samples submitted for PNH/ FCM screening for the past 2 years (2012-2013) at hematology section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center (General Organization). We collected the positive cases and reviewed them for age, gender, indication for screening, sample type, size of the PNH clone and cell type affected. Immunophenotypic analysis was performed using gating antibodies CD45, CD15, CD33, CD235a GPI-linked antibodies, CD59, CD14, and CD24 as well as fluorescent Aerolysin (FLAER). In a total of 366 peripheral blood samples submitted for PNH/ FCM screening fifteen samples (4%) were positive for PNH clones but only 12 patients were available for analysis. The median age for our patients was 34 years with approximately equal male to female distribution. 12 cases showed type II and III clones within the RBCs with clone size ranging between 0.04% and 56%. Analysis of granulocytes and monocytes revealed type III clone in 8 cases, type II and III clone in 3 cases and non in one case. The percentage of the clone varies between the granulocytes and monocytes and ranges from 1% up to 100%. Of 12 positive PNH cases, 8 (66.7%) patients were diagnosed as having aplastic anemia (AA), 1 (8.3%) patient with Budd-Chiari syndrome, 1 (8.3%) patient has chronic immune thrombocytopenia (ITP), and 2 (16.7%) patients presented with pancytopenia. This study confirms the rarity of the disease since only 4% of the submitted samples for analysis turned to be positive for PNH. The detection limit for a PNH clone by FCM in the RBC or WBC is 0.01%. Identification of small PNH clone is greater FCM sensitivity relative to old test used for the same purpose (Ham test). The use of the FLAER allowed us to detect granulocytic PNH clone, however, granulocytes PNH clone detection alone without RBCs clone detection is not recommended. This review confirms the previous percentage of positive cases (5.9%) reported from this center on a smaller number of cases during the past few years. Disclosures No relevant conflicts of interest to declare.
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Gautam, Pratiksha, and Hemraj Saini. "A Mutation Operator-Based Scenario for Evaluating Software Clone Detection Tools and Techniques." International Journal of Information Security and Privacy 13, no. 1 (January 2019): 30–45. http://dx.doi.org/10.4018/ijisp.2019010103.
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Over the past few years, several software clone detection tools and techniques have been introduced by numerous researchers. The software clone detection techniques and tools are based on their numerous attributes and sub-attributes which make them difficult to complete a comparative study. Therefore, the authors propose a mutation operator-based editing taxonomy for generating different software clone types. In addition, a hypothetical scenario is developed using mutation operator-based editing taxonomy and this hypothetical scenario is used to evaluate various software clone detection techniques and tools. Further, the existing evaluation criterion is extended by the hypothetical scenario which is clearly represented by the analysis of results.
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Vosberg, Sebastian, Luise Hartmann, Stephanie Schneider, Klaus H. Metzeler, Bianka Ksienzyk, Kathrin Bräundl, Martin Neumann, et al. "Detection of Chromosomal Aberrations in Acute Myeloid Leukemia By Copy Number Alteration Analysis of Exome Sequencing Data." Blood 126, no. 23 (December 3, 2015): 3859. http://dx.doi.org/10.1182/blood.v126.23.3859.3859.
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Abstract Exome sequencing is widely used and established to detect tumor-specific sequence variants such as point mutations and small insertions/deletions. Beyond single nucleotide resolution, sequencing data can also be used to identify changes in sequence coverage between samples enabling the detection of copy number alterations (CNAs). Somatic CNAs represent gain or loss of genomic material in tumor cells like aneuploidies (e.g. monosomies and trisomies), duplications, or deletions. In order to test the feasibility of somatic CNA detection from exome data, we analyzed 13 acute myeloid leukemia (AML) patients with known cytogenetic alterations detected at diagnosis (n=8) and/or at relapse (n=11). Corresponding remission exomes from all patients were available as germline controls resulting in 19 comparisons of paired leukemia and remission exome data sets. Exome sequencing was performed on a HiSeq 2500 instrument (Illumina) with mean target coverage of >100x. Exons with divergent coverage were detected using a linear regression model on mean exon coverage, and CNAs were called by an exact segmentation algorithm (Rigaill et al. 2012, Bioinformatics). For all samples, cytogenetic information was available either form routine chromosomal analysis or fluorescent in situ hybridization (FISH). Blast count were known for all but one AML sample (n=19). Copy number-neutral cytogenetic alterations such as balanced translocations were excluded from the comparative analysis. By CNA-analysis of exomes we were able to detect chromosomal aberrations consistent with routine cytogenetics in 18 out of 19 (95%) AML samples. In particular, we confirmed 2 out of 2 monosomies (both -7), and 9 out of 10 trisomies (+4, n=1; +8, n=8; +21, n=1), e.g. trisomy 8 in figure 1A. Partial amplifications or deletions of chromosomes were confirmed in 10 out of 10 AML samples (dup(1q), n=3; dup(8q), n=1; del(5q), n=3; del(17p), n=1; del(20q), n=2), e.g. del(5q) in figure 1B. In the one case with inconsistent findings of chromosomal aberrations between exome and cytogenetic data there was a small subclone harboring the alteration described in only 4 out of 21 metaphases (19%). To assess the specificity of our CNA approach, we analyzed the exomes of 44 cytogenetically normal (CN) AML samples. Here we did not detect any CNAs larger than 5 Mb in the vast majority of these samples (43/44, 98%), only one large CNA was detected indicating a trisomy 8. Estimates of the clone size were highly correlated between CNA-analysis of exomes and the parameters from cytogenetics and cytomorphology (p=0.0076, Fisher's exact test, Figure 1C). In CNA-analysis of exomes, we defined the clone size based on the coverage ratio: . Clone size estimation by cytogenetics and cytomorphology was performed by calculating the mean of blast count and abnormal metaphase/interphase count. Of note, clones estimated by CNA-analysis of exomes tended to be slightly larger. This may result from purification by Ficoll gradient centrifugation prior to DNA extraction for sequencing and/or the fact that the fraction of cells analyzed by cytogenetics does not represent the true size of the malignant clone accurately because of differences in the mitotic index between normal and malignant cells. Overall, there was a high correlation between our CNA analysis of exome sequencing data and routine cytogenetics including limitations in the detection of small subclones. Our results confirm that high throughput sequencing is a versatile, valuable, and robust method to detect chromosomal changes resulting in copy number alterations in AML with high specificity and sensitivity (98% and 95%, respectively). Figure 1. (A) Detection of trisomy 8 with an estimated clone size of 100% (B) Detection of deletion on chromosome 5q with an estimated clone size of 90% (C) Correlation of clone size estimation by routine diagnostics and exome sequencing (p=0.0076) Figure 1. (A) Detection of trisomy 8 with an estimated clone size of 100%. / (B) Detection of deletion on chromosome 5q with an estimated clone size of 90%. / (C) Correlation of clone size estimation by routine diagnostics and exome sequencing (p=0.0076) Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
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Williamson, John, Timothy Looney, Geoffrey Lowman, Harwinder Sidhu, Luis Solano, Suzanne Salazar, Huan Tian, and Rajendra Ramsamooj. "Detection of Low Frequency T-Cell Clones Via a Next Generation Sequencing (NGS) Based Immune-Oncology Research Assay." Blood 134, Supplement_1 (November 13, 2019): 5774. http://dx.doi.org/10.1182/blood-2019-127459.
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Introduction: The ability to detect and quantitate low frequency T-cell clones enables numerous hematology/oncology research applications, including identification and assessment of biomarkers associated minimal residual disease (MRD). Rare clone detection via NGS requires highly efficient library preparation and accurate sequencing methodologies, because single nucleotide substitution sequencing errors mimic the natural variation in the T-cell repertoire, resulting in detection of artifactual low frequency clones. Here we present an experimental framework and corresponding performance of rare clone detection utilizing the OncomineTM TCR Beta short read (TCRb-SR) assay, using Ampliseq-based library preparation targeting the highly variable CDR3 region of TCRb using either DNA or RNA as input, with sample-to-result in 2 days. Methods: To evaluate detection sensitivity of the TCRB-SR assay, we utilized Jurkat cell line DNA and RNA because the presence of a single T cell clone enables precise control of dilution studies. Commercially procured Jurkat gDNA or RNA was spiked into peripheral blood leukocyte gDNA or RNA from 10-1 to 10-6 absolute clone frequency to create specimens with a known T-cell clone at frequencies commonly observed in MRD research applications. Peripheral blood leukocyte gDNA or RNA was used as the background for spike in studies due to its high T-Cell diversity. Six to twenty technical replicates were analyzed per dilution point, with DNA inputs ranging from 100ng to 1ug and RNA inputs ranging from 25ng to 100ng to evaluate the minimum detectable clone frequency as a function of nucleic acid input. Libraries were prepared following the TCRB-SR manufacturer's instructions for both DNA and RNA, followed by templating and sequencing using Ion Chef and S5 systems. Data processing was performed in Torrent Suite software (v5.10) followed by read alignment to the IMGT database of variable, diversity, and joining genes using Ion Reporter software (v5.10). For the 10-6 target frequency with gDNA as the input, four 1ug libraries were combined for analysis in Ion Reporter. Analytical sensitivity was calculated at each target clone frequency by detection of the Jurkat clone as defined by V-gene, Joining gene, and CDR3 nucleotide sequence. Results: Detection sensitivity was dependent on the amount gDNA or RNA input. For gDNA inputs, we observed 100% sensitivity at 10-3 with 100ng input, 100% sensitivity at 10-4 with 250ng input, 95% sensitivity at 10-5 with 1ug input, and 100% sensitivity at 10-6 with 4ug input. For RNA inputs, we observed 100% sensitivity at 10-5 with 25ng input and 100% sensitivity at 10-6 with 100ng input. In addition, we observe a strong linearity of observed clone frequencies at each dilution level, with an r-squared of 0.97. Conclusions: Here we demonstrate the ability to detect T-cell clones down to 10-6 from gDNA or RNA inputs with high sensitivity and linearity utilizing the OncomineTM TCR Beta short read assay. We present data demonstrating detection of clones with absolute frequencies of 10-6 utilizing 4ug gDNA input or 100ng RNA input, highlighting strong performance at nucleic acid input levels typically seen in clinical research samples. Taken together, we show feasibility for rare clone detection in either gDNA or RNA enabling research and development for T-cell minimal residual disease applications. For research use only, not for use in diagnostic procedures. Disclosures Williamson: Thermo Fisher Scientific: Employment. Looney:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Sidhu:Thermo Fisher Scientific: Employment. Solano:Thermo Fisher Scientific: Employment. Salazar:Thermo Fisher Scientific: Employment. Tian:Thermo Fisher Scientific: Employment. Ramsamooj:Thermo Fisher Scientific: Consultancy.
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Khan, Wazir Zada, Md Shohrab Hossain, Mohammed Y. Aalsalem, N. M. Saad, and Mohammed Atiquzzaman. "A cost analysis framework for claimer reporter witness based clone detection schemes in WSNs." Journal of Network and Computer Applications 63 (March 2016): 68–85. http://dx.doi.org/10.1016/j.jnca.2016.01.014.
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Alalfi, Manar H., Elizabeth P. Antony, and James R. Cordy. "An approach to clone detection in sequence diagrams and its application to security analysis." Software & Systems Modeling 17, no. 4 (September 12, 2016): 1287–309. http://dx.doi.org/10.1007/s10270-016-0557-6.
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Xiu, Leshan, Chi Zhang, Yamei Li, Feng Wang, and Junping Peng. "High-resolution melting analysis for rapid detection of the internationally spreading ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone." Journal of Antimicrobial Chemotherapy 75, no. 1 (September 17, 2019): 106–9. http://dx.doi.org/10.1093/jac/dkz395.
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Abstract Objectives Increased awareness of the international spread of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone, which threatens recommended dual therapy, is essential. The objective of the present study was to develop and evaluate a rapid, simple and cost-effective method based on high-resolution melting (HRM) analysis for direct detection of the FC428 clone from clinical isolates and specimens. Methods The singleplex HRM assay was designed to identify the FC428 clone by using specific primers, which flank the alteration A311V in the penA-60.001 allele. Analytical performance was initially evaluated by testing 623 isolates and a panel of non-gonococcal strains. To ensure the method can be directly applied in clinical samples, two internal control targets (opa and porA) were also designed and included in the final multiplex HRM assay. Two hundred and eighty-two clinical samples (94 urine and 188 urethral/genital swabs) were then analysed using this multiplex HRM assay. Results The FC428 clone was easily differentiated from the non-mosaic alleles and other mosaic alleles without A311 mutations by comparing the differences in melt curves. Cross-reactivity was not observed for the penA-60.001 allele when testing 15 non-gonococcal Neisseria strains. When applied to the 623 isolates, the HRM assay successfully characterized one isolate as an FC428 clone (MLST1903, NG-MAST3435, NG-STAR233). Our data show that the multiplex HRM assay with high specificity can be directly applied in clinical samples. Conclusions This method can generate results within 90 min at a cost of less than US$0.5 per isolate or sample, making this assay an ideal tool for large epidemiological studies to enhance surveillance of the internationally transmitted ceftriaxone-resistant N. gonorrhoeae FC428 clone.
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Correia, Rodolfo Patussi, Laiz Cameirão Bento, Ana Carolina Apelle Bortolucci, Anderson Marega Alexandre, Andressa da Costa Vaz, Daniela Schimidell, Eduardo de Carvalho Pedro, et al. "Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria." Einstein (São Paulo) 14, no. 3 (September 2016): 366–73. http://dx.doi.org/10.1590/s1679-45082016ao3641.
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ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.
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O’Keefe, Christine L., Alexander Rodriguez, Evan Howe, and Jaroslaw P. Maciejewski. "Differential Comparative Genomic Hybridization Analysis of Normal and Glycosyl Phosphatidyl Inositol Deficient Clones in Paroxysmal Noctorunal Hemoglobinuria." Blood 104, no. 11 (November 16, 2004): 2831. http://dx.doi.org/10.1182/blood.v104.11.2831.2831.
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Abstract Various theories can explain the expansion of glycosyl phosphatidyl inositol anchored protein (GPI-AP)-deficient clones in PNH. The differential growth advantage of PIG-A mutant stem cell scould be due to immune escape in the context of an autoimmune attack on hematopoietic targets explaining the close clinical relationship to aplastic anemia. The causes of evolution of the PNH clone may be related to a less efficient immune recognition of PNH cells, their relative resistance to apoptosis or slower senescense. These changes may be related to additional mutations or genetic damage present in either normal or GPI-AP deficient hematopoietic clone. Using traditional karyotyping, chromosomal defects are not common in PNH but the limited resolution of this method may preclude detection of smaller aberrations. Due to these limitations, novel technologies which improve resolution and sensitivity are under development. In array-based comparative genomic hybridization (A-CGH), differentially labeled test and reference DNA samples are hybridized to genomic microarrays. Differences in sequence copy number between the samples are reflected in a shift of the fluorescence spectrum. The principle of A-CGH allows for detection of unbalanced chromosomal changes of the whole genome and its resolution is limited solely by the number of clones. In preliminary studies we found significantly decreased telomerase activity in normal vs. CD59−CD55− CD34+ cells derived from patients with PNH suggesting: 1) more accelerated telomere shortening in phenotypically normal hematopoietic cells derived from PNH patients, or 2) the possibility of chromosomal instability and acquisition of karyotypic defects should a critical telomere length be reached. We hypothesized that higher resolution analysis of the karyotypic changes in PNH using CGH may result in detection of cryptic karyotypic abnormalities present in either the normal or GPI-AP deficient clone. We have analyzed a cohort of patients (N=6) with hemolytic PNH and major expansion of a GPI-AP deficient clone (50–95% of PNH granulocytes). All patients had normal cytogenetics by metaphase karyotyping. We have separated GPI-AP-deficient myeloid peripheral blood cells using CD55 and CD59 as well as CD2 and CD19 staining to exclude lymphocytes. Separated CD55−CD59− and CD55+CD59+ non lymphoid cells were sorted and, following DNA extraction, subjected to A-CGH analysis (Vysis microarrays containing 287 probes). In one patient, PNH cells appeared normal by A-CGH but the corresponding “normal” cells contained deletions of telomeric region of several chromosomes (2, 5, 16, 18, 19, 20qtel as well as 5, 11,19ptel). However, in the remaining patients telomeric deletions were present in both normal and PNH cells. There was no difference in the numbers of chromosomes affected and diverse chromosomal telomeric deletions were present. No other lesions were found. By comparison when A-CGH analysis was performed in 8 normal individuals telomeric deletions were only rarely found and only occasional gains of contiguous clones on chromosomes were detected. Deletion of the telomeric portions of multiple chromosomes is compatible with accelerated global telomere loss in PNH. However, it appears that this phenomenon was not restricted to either normal or PNH cells. Our results are in agreement with previously described telomere shortening measured by FISH, flow cytometry or Southern blot analysis in PNH, a change not consistently restricted to normal cells.
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Vass, M., L. Kotkova, I. Diblikova, Z. Nevorankova, Cooper KM, Kennedy DG, and M. Franek. "Production and characterisation of monoclonal antibodies for the detection of AOZ, a tissue bound metabolite of furazolidone." Veterinární Medicína 50, No. 7 (March 28, 2012): 300–310. http://dx.doi.org/10.17221/5627-vetmed.
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3-amino-2-oxazolidinone (AOZ) is a tissue bound toxic metabolite derived from the nitrofuran antibiotic, furazolidone. AOZ is detected in the derivatised form of 3-{[(2-nitrophenyl) methylene] amino}-2-oxa-zolidinone (NP AOZ). 3-{[(3-carboxyphenyl)-methylene] amino-2-oxazolidinone (CP AOZ) was used as the immunising hapten for the production of monoclonal antibodies against NP AOZ. Monoclonal antibodies were produced using hybridomas from the fusion of murine myeloma cells and spleen cells isolated from BALB/c mice immunised with CP AOZ-ethylenediamine-human serum albumin (CP AOZ-ed-HSA). The antibody production in ascitic fluids from clones 3B8/2B9 and 2D11/A4 was monitored during a 16 month period. Repeated cultures of these hybridomas, followed by injection into mice and cloning did not change the assay parameters. Clone 2D11/A4 exhibited long term stability in antibody production throughout the experiment whereas clone 3B8/2B9 demonstrated variability in particular antibody yields whilst retaining assay sensitivity. Reasons for this production variability in clones are discussed. In an optimised direct ELISA format, the antibodies exhibited a 50% binding inhibition in the range of 0.52–1.15 ng/ml with NP AOZ (0.22 -0.50 ng/ml, respective AOZ equivalents) and showed high specificity towards this analyte. The sensitivity of monoclonal antibodies incorporated into the ELISA is compatible with the European Union MRLP and is currently in use for routine analysis.
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Kim, G. D., I. C. Carr, and G. Milligan. "Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and β2-adrenoceptor-expressing NG108-15 cells." Biochemical Journal 308, no. 1 (May 15, 1995): 275–81. http://dx.doi.org/10.1042/bj3080275.
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Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha-subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2-adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose-effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost-stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor.
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Inagaki, Ryosaku, Masahiro Marshall Nakagawa, Yasuhito Nannya, Qi Xingxing, Lanying Zhao, June Takeda, Akinori Yoda, et al. "Analysis of Mechanisms Underlying Clonal Evolution of AML By a New Single-Cell Sequencing Platform." Blood 134, Supplement_1 (November 13, 2019): 542. http://dx.doi.org/10.1182/blood-2019-126444.
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Background Leukemic cell populations are highly heterogeneous in terms of both gene mutations and gene expression, which is shaped by acquisition of multiple mutations and expansion of adapted clone. This evolutional process is clinically important because it is observed in the contexts of treatment resistance and relapse as well as leukemic transformation, and molecular mechanisms involved in clonal selection can be exploited as a therapeutic target. Nevertheless, direct analysis of such mechanisms in patients' cells is hampered by technical difficulties to characterize both clonal structure and gene expression at a single-cell resolution. On this issue, we have recently developed a new method which enables simultaneously detection of mutations and whole transcriptome information at single-cell level by extensively modifying an existing single cell RNA-seq (Nakagawa et al. ASH abstract 2018). The aim of this study is to understand heterogeneity of clones and to clarify mechanisms behind clonal expansion in AML by longitudinal analysis using our novel single-cell sequencing platform. Results In order to estimate clone frequencies and select samples to be analyzed by single-cell sequencing, we first sequenced bulk bone marrow cells from patients with AML. Of interest, we found that AML samples frequently harbored multiple clones having different Ras pathway mutations, most frequently involving NRAS, which exhibited dynamic change in their clone size during the course of AML. These are interesting targets of the analysis of mechanism of clonal evolution of AML. Thus, three patients having multiple (n=3-5) Ras pathway mutations were investigated by sequencing their bone marrow Lin-, CD34+ cells using the newly established single-cell method, which successfully separated distinct clones having distinct mutations, where all of detected Ras pathway mutations were present in independent clones as expected. In order to examine these independent clones with Ras pathway mutations might show equal or heterogenous cellular phenotypes, proliferation or differentiation statuses as determined from transcriptome data was analyzed for all detected NRAS mutated clones. Among the NRAS mutated clones, some showed significant increase in proliferation-associated gene expression signature (calculated as proliferation score) compared with NRAS wild type clones, and no NRAS mutated clones showed decrease of the score, which is consistent with pro-proliferative function of Ras pathway. Interestingly, such increase in proliferation showed considerable heterogeneity among clones, where some NRAS mutated clones showed greatly increased proliferation scores compared to other NRAS mutated clones. Differentiation statuses of NRAS clones also showed heterogeneity among clones. In order to examine whether this inter-clone proliferation difference correlates with clone dynamics, we then analyzed longitudinal bone marrow samples for a patient who showed different proliferation between clones. The NRAS mutated clone with highly increased proliferation compared with wild type clone (NRAS p.G12S) had undergone rapid expansion in 3 months (cell frequency 0.08 to 0.74) in spite of continuous azacitidine treatment, while the NRAS mutated clone with less increase in proliferation (NRAS p.G12D) had showed regression (cell frequency 0.72 to 0.14). To investigate the mechanism of this therapy-resistant clonal expansion, we compared transcriptome data of these clones. Unlike the regressed clone, the expanded clone uniquely exhibited increase in expression of genes in PI3K/AKT pathway and unfolded protein response (UPR) pathway, one of cellular stress response pathway. UPR is recently reported to responsible for the promoted survival and competitive advantage in mouse hematopoietic stem cells with Nras mutations (Liu et al. Nat. Cell Biol. 2019). Our data suggest that the enhanced UPR pathway contributes to clonal expansion also in human AML with Ras pathway mutations. Conclusions Using a newly developed single-cell sequencing platform, we have successfully characterized gene expression profiles associated with clonal evolution of AML with Ras pathway mutations. Simultaneous measurement of both mutations and transcriptomes at a single-cell level will help understand the mechanism of clonal evolution of AML. Disclosures Inagaki: Sumitomo Dainippon Pharma Co., Ltd.: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Yoda:Chordia Therapeutics Inc.: Research Funding. Ogawa:RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy.
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Schönland, Stefan, Ute Hegenbart, Christoph Kimmich, Katarina Lisenko, Dirk Hose, Hartmut Goldschmidt, Anna Jauch, Anthony D. Ho, and Michael Hundemer. "Detection and Characterization of Plasma Cell and B Cell Clones in Patients with Systemic Light Chain Amyloidosis Using Flow Cytometry." Blood 124, no. 21 (December 6, 2014): 2068. http://dx.doi.org/10.1182/blood.v124.21.2068.2068.
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Abstract Introduction: AL amyloidosis is a rare and life-threatening protein-deposition disorder caused by a small B cell (mostly plasma cell) clone which produces amyloidogenic light chains. The goal of therapy is to target this clone and halt the uncontrolled release of free light chain, which might subsequently lead to improvement of organ function. In routine diagnostic some of these B cell clones are missed as they might be extremely small. However, specific treatment can only be applied if the clone is well characterized. Hardly any data on the characteristics of these cells using flow cytometry have been reported. (e.g. Paiva et al., Blood 2011). Study design: We performed a retrospective analysis of consecutive patients who were referred to our amyloidosis center (March to July 2014) and have been thoroughly studied (immunhistology of amyloid, free light chain assay, immunofixation, bone marrow diagnostic: cytology, flow cytometry and interphase-FISH cytogenetics (iFISH)). Patients and Methods: Twenty-two patients were included (all untreated, 21 AL patients, one pt with monoclonal gammopathy of renal significance (MGRS)). Plasma cells were detected by their co-expression of CD38 and CD138 antigens. Differentiation between malignant and normal plasma cells was achieved by analysis of aberrant CD45 and CD19 expressions and proof of intracellular light chain restriction (see Figure 1). To evaluate potential targets for an antibody-based immunotherapy, we stained CD20, CD22, CD30, CD52 and CS-1 on these plasma cells. Overall, positivity was defined as >20% expression of the antigen. iFISH was done after CD138 selection as previously described (Bochtler et al., Blood 2011). Results: Main characteristics and results are shown in Table 1. Median dFLC was 304 mg/l, three patients had a dFLC of less than 50 mg/l. Median plasma cell count in cytology was 10%, 3 patients had less than 5%. Median plasma cell count by flow was 3.8%, three patients had less than 1%. Correlation between dFLC, plasma cell count in cytology and flow was low (FLC vs. flow: spearman=0.25, p=0.26; FLC vs. cytology: spearman=0.49, p=0.02; flow vs. cytology, spearman=0.36, p=0.1). Detection of the amyloidogenic clone by flow was possible in all but one patient (95%). In this patient we were not able to show a light chain restriction although we detected a relevant aberrant plasma cell clone (CD45low, CD19low). In one patient we found a B cell lymphoma as underlying disease for MGRS type IgG lambda (CD19+, CD20+, lambda+, CD5-, CD22+, FMC7-, CD23-, CD25+, CD103-, CD38+ typical for marginal zone lymphoma). In all 21 patients the light chain restriction demonstrated by flow was confirmed by immunofixation, FLC, and immunohistology of the amyloid. All patients analyzed for the expression of CS-1 were positive. 25% were also positive for CD20 and none was positive for CD22, CD30 and CD52. Detection of the plasma cell clone by iFISH was possible in all 21 patients (see Table 1). Conclusion: Flow cytometric analysis of the bone marrow is a very sensitive method to detect and characterize the amyloidogenic clone in AL amyloidosis. B cell lymphomas can easily be distinguished from pure plasma cell clones. Secondly, flow provides useful information to specify immune-chemotherapy in AL amyloidosis and related disorders. Table 1: Patients (n=22) Characteristics and Results Age in yrs (median / range) 67 (41 – 77) Sex: female / male 9 / 13 Type of light chain: kappa / lambda 4 / 18 Median dFLC in mg/l (range) 304 (22 - 6621) Median % of plasma cells in BM cytology (range) 10 (0 – 68) Underlying disease leading to AL amyloidosis“MG” / MM III / B-NHL 20 / 1 / 1 Median % of PC by flow (range) 3.8 (0.2 - 34) Detection of the amyloidogenic clone by flow 21 / 22 Flow analysis of clonal plasma cells (% of pts)CD20+ / CD22+ / CD30+ / CD52+ / CD56+ / CS-1+ 25 / 0 / 0 / 0 / 75 / 100 Detection of a clone by iFISH 21/21 % of pts with t(11;14) / Gain of 1q21 / Hyperdiploidy / High-risk cytogenetic (del 17p13, t(4;14)) 52 / 10 / 14 / 10 Figure 1: Representative flow analysis of one pt. with a lambda+, CD38+, CD138+ plasma cell clone (green). Polyclonal CD19+ B cells in red. Figure 1:. Representative flow analysis of one pt. with a lambda+, CD38+, CD138+ plasma cell clone (green). Polyclonal CD19+ B cells in red. Disclosures Schönland: Janssen: Honoraria; Celgene: Honoraria. Hegenbart:Janssen: Honoraria; Celgene: Honoraria. Hose:Novartis: Research Funding. Hundemer:Celgene: Honoraria, Research Funding.
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TAKAHASHI, Masakazu, Reiji NANBA, Yunarso ANANG, and Yoshimichi WATANABE. "An Improvement Method for Program Structure Using Code Clone Detection, Impact Analysis, and Refactoring Formats." SICE Journal of Control, Measurement, and System Integration 10, no. 3 (2017): 184–91. http://dx.doi.org/10.9746/jcmsi.10.184.
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Mao, Yiping, and Marion Cremer. "Detection of Duchenne muscular dystrophy carriers by dosage analysis using the DMD cDNA clone 8." Human Genetics 81, no. 2 (January 1989): 193–95. http://dx.doi.org/10.1007/bf00293903.
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Tekchandani, Rajkumar, Rajesh Bhatia, and Maninder Singh. "Semantic code clone detection for Internet of Things applications using reaching definition and liveness analysis." Journal of Supercomputing 74, no. 9 (August 6, 2016): 4199–226. http://dx.doi.org/10.1007/s11227-016-1832-6.
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Xie, Jiahua, Todd C. Wehner, and Mark A. Conkling. "PCR-based Single-strand Conformation Polymorphism (SSCP) Analysis to Clone Nine Aquaporin Genes in Cucumber." Journal of the American Society for Horticultural Science 127, no. 6 (November 2002): 925–30. http://dx.doi.org/10.21273/jashs.127.6.925.
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Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes.
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Fotirić Akšić, Gašić, Dabić Zagorac, Sredojević, Tosti, Natić, and Meland. "Chemical Fingerprint of ‘Oblačinska’ Sour Cherry (Prunus cerasus L.) Pollen." Biomolecules 9, no. 9 (August 21, 2019): 391. http://dx.doi.org/10.3390/biom9090391.
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The aim of this research was to analyze sugars and phenolics of pollen obtained from 15 different ‘Oblačinska’ sour cherry clones and to assess the chemical fingerprint of this cultivar. Carbohydrate analysis was done using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD), while polyphenols were analyzed by ultra-high-performance liquid chromatography–diode array detector–tandem mass spectrometry (UHPLC-DAD MS/MS) system. Glucose was the most abundant sugar, followed by fructose and sucrose. Some samples had high level of stress sugars, especially trehalose. Rutin was predominantly polyphenol in a quantity up to 181.12 mg/kg (clone III/9), with chlorogenic acid (up to 59.93 mg/kg in clone III/9) and p-coumaric acid (up to 53.99 mg/kg in clone VIII/1) coming after. According to the principal component analysis (PCA), fructose, maltose, maltotriose, sorbitol, and trehalose were the most important sugars in separating pollen samples. PCA showed splitting off clones VIII/1, IV/8, III/9, and V/P according to the quantity of phenolics and dissimilar profiles. Large differences in chemical composition of studied ‘Oblačinska sour cherry’ clone pollen were shown, proving that it is not a cultivar, but population. Finally, due to the highest level of phenolics, clones IV/8, XV/3, and VIII/1 could be singled out as a promising one for producing functional food and/or in medicinal treatments.
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Luo, Zhenhao, Baosheng Wang, Yong Tang, and Wei Xie. "Semantic-Based Representation Binary Clone Detection for Cross-Architectures in the Internet of Things." Applied Sciences 9, no. 16 (August 10, 2019): 3283. http://dx.doi.org/10.3390/app9163283.
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Code reuse is widespread in software development as well as internet of things (IoT) devices. However, code reuse introduces many problems, e.g., software plagiarism and known vulnerabilities. Solving these problems requires extensive manual reverse analysis. Fortunately, binary clone detection can help analysts mitigate manual work by matching reusable code and known parts. However, many binary clone detection methods are not robust to various compiler optimization options and different architectures. While some clone detection methods can be applied across different architectures, they rely on manual features based on human prior knowledge to generate feature vectors for assembly functions and fail to consider the internal associations between features from a semantic perspective. To address this problem, we propose and implement a prototype GeneDiff, a semantic-based representation binary clone detection approach for cross-architectures. GeneDiff utilizes a representation model based on natural language processing (NLP) to generate high-dimensional numeric vectors for each function based on the Valgrind intermediate representation (VEX) representation. This is the first work that translates assembly instructions into an intermediate representation and uses a semantic representation model to implement clone detection for cross-architectures. GeneDiff is robust to various compiler optimization options and different architectures. Compared to approaches using symbolic execution, GeneDiff is significantly more efficient and accurate. The area under the curve (AUC) of the receiver operating characteristic (ROC) of GeneDiff reaches 92.35%, which is considerably higher than the approaches that use symbolic execution. Extensive experiments indicate that GeneDiff can detect similarity with high accuracy even when the code has been compiled with different optimization options and targeted to different architectures. We also use real-world IoT firmware across different architectures as targets, therein proving the practicality of GeneDiff in being able to detect known vulnerabilities.
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Martin-Cabrera, Pedro, Claudia Haferlach, Torsten Haferlach, Wolfgang Kern, and Susanne Schnittger. "Co-Occurrence of Separate BCR-ABL1-Positve and JAK2V617F-Positive Clones in 23 Patients Reveals Biologic and Clinical Importance." Blood 124, no. 21 (December 6, 2014): 3175. http://dx.doi.org/10.1182/blood.v124.21.3175.3175.
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Abstract Background: The simultaneous detection of a BCR-ABL1 rearrangement and a JAK2V617F mutation in the same patient is a very rare event and has previously been described in case reports or very small series of cases only. Aim: 1) To establish the incidence of cases with concurrent BCR-ABL1 rearrangement and JAK2V617F mutation. 2) Evaluate whether one clone harbours both mutations or whether there are two independent clones. 3) Establish whether these patients have additional concurrent gene mutations and how they influence the evolution of both diseases. Patients and Methods: A total of 27,907 patients with suspected myeloproliferative neoplasms (MPN) where studied in parallel for BCR-ABL1 and JAK2V617F mutation from May 2005 to June 2014 at our institution. BCR-ABL1 analysis was performed by multiplex RT-PCR and JAK2V617F mutation analysis by melting curve based LightCycler assay. A total of 23 patients (0.08%) were positive for both mutations. Eleven patients were male and 12 were female with a median age at diagnosis of 72 years (range 46-80 years). Of fifteen patients 2 or more sample time points were available for follow-up analyses (median follow-up: 4 years, range: 5 months - 9 years). Both BCR-ABL1 and JAK2V617F mutation loads were assessed by quantitative real time PCR. In addition, 22/23 cases were analyzed upon detection of co-occurrence of both clones with a pan-myeloid gene panel consisting of 25 genes: TET2, RUNX1, PHF6, ASXL1, CBL, DNMT3A, SF3B1, TP53, BCOR, BRAF, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, KIT, KRAS, MPL, NPM1, NRAS, SRSF2, U2AF1, and WT1. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sequence NGS platform (Roche 454, Branford, CT). The median coverage per amplicon was 2,215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%. Results: At the time point of detection of both mutations morphological assessment was available in 12 patients. The remaining 5 showed features typical for CML. Bone marrow blast count was <5% in all cases. Cytogenetics was available in 18/23 cases (78.3%). The classical t(9;22)(q34;q11) was identifiable in 16/18 patients. Two patients had a normal karyotype as they were in complete cytogenetic remission of their CML (due to TKI treatment) at diagnosis of the JAK2 V617F positive clone. In the majority of patients (n=16) the JAK2V617F mutation predated the BCR-ABL1 clone, in 4 patients CML was known before the detection of the JAK2V617 positive clone, in 1 patient both were diagnosed simultaneously and in another 2 patients information in this regard was lacking. BCR-ABL1 transcript types distributed as follows: b2a2 and/or b3a2 (n=18), and e1a2 (n=5). The continuous quantitative assessment of BCR-ABL1 and JAK2V617F mutational loads in 15 patients showed asynchronous patterns of courses in all cases giving proof of these aberrations representing two different clones in these cases. When treatment with TKI was initiated, the BCR-ABL1 clone decreased while the JAK2V617F clone either remained stable or increased in all 15 cases. Next generation sequencing revealed further mutations in 13/22 analyzed patients (56.5%). One mutation was detected in 8 patients, 4 patients revealed 2, and one patient even 3 different additional mutations. In detail, mutations in the following genes were detected: TET2 (n=8), ASXL1 (n=4), RUNX1 (n=2), CBL (n=1), DNMT3A (n=1), PHF6 (n=1) SF3B1 (n=1) and TP53 (n=1). These mutations were traced and quantified retrospectively. Data suggests that they were most probably present in the JAK2V617F positive clone. This again supports the theory of both clones being independent of each other. Conclusions: 1) Co-occurrence of BCR-ABL1 and JAK2V617F is a very rare event (0.08%). 2) BCR-ABL1 and JAK2V617F represent two different clones. 3) Additional gene mutations are detected in 56% of these cases and all seem to be within the JAK2V617F positive clone. 4) Clinically, the BCR-ABL1 clone is easily controlled with TKI, however, the combined management of the JAK2V617F clone is more challenging especially when a fibrotic phase of the disease takes over. The long-term effect of JAK2-inhibitors in the management of these patients is yet to be established. Disclosures Martin-Cabrera: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Kwoun, Woo Jae, Jeong-Yeal Ahn, Ja Young Seo, Jae Hoon Lee, Hawk Kim, Jinny Park, Kwai Han Yoo, Sung-Ran Cho, and Hye Ryoun Kim. "Evaluation of Clinical Usefulness of Monocyte Gating Using CD14 and CD64 for Detecting PNH Clone By Flow Cytometry." Blood 132, Supplement 1 (November 29, 2018): 4947. http://dx.doi.org/10.1182/blood-2018-99-111468.
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Abstract Introduction Flow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)-linked protein expression on red blood cell, granulocyte, and monocyte. The current assays are 4-color analyses of GPI-linked markers such as fluorescein-labeled proaerolysin (FLAER), CD24, CD14, CD59, and CD235a and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. We investigated the utility of CD14/CD64 monocyte gating by comparing with CD45/light scatter (LS) gating in PNH study of the patients with cytopenia and analyzed the types and cell lineages of PNH clone according to the disease groups. Method Total 138 cases were recruited in this study from July 2017 to February 2018 at Gachon University Gil Medical Center in Korea. Flow cytometric analysis was performed with EDTA blood by Beckman Coulter Cytomics FC500 cytometer using gating antibodies such as CD45, CD14, CD15, CD64, CD235a and GPI-linked antibodies such as CD59, CD14, CD24, FLAER. The proportion of monocyte was estimated by CD14/CD64 gating and compared with those using CD45/LS gating. The type of PNH clone was defined according to the size of PNH population. A PNH clone is defined as a PNH population exceeding 1% of the gated cells, a minor PNH clone as between 0.1 and 1%, and rare cells with GPI-deficiency defined as a PNH population less than 0.1%. The types and cell lineages of the PNH clone were analyzed according to the disease groups. Statistical analysis was done using SPSS 17.0 and MedCalc 15.2, and P<0.05 was considered statistically significant. Results Of the 138 cases, PNH clone was detected with 27 cases including 15 cases with a PNH clone and 12 cases with a minor PNH clone. PNH clone was observed in all 8 cases (100%) of PNH cases. Two PNH clone and 4 minor PNH clones were identified in 6 of 16 cases (38%) of acute myeloid leukemia. In 6 of 21 cases (29%) of aplastic anemia (AA) show 5 PNH clones and 1 minor PNH clone. In 5 of 78 cases (6%) of cytopenia(s) only minor PNH clone was observed. The CD45 plus LS gating in monocyte represents a sensitivity of 100%, a specificity of 40.2%, and 60% (73/89) false positive rate in detecting of PNH clone. McNemar test indicates a significant difference between CD14/CD64 and CD45/LS gating methods (P = 0.00). The Bland-Altman plot of monocyte proportion between the two gating methods revealed that CD45/LS gating method was tended to underestimate monocyte proportion and the larger the number of monocytes, the greater the difference in number of monocyte between the two gating methods. The trend of the size of PNH clone in each cell lineage was confirmed by follow-up in three patients with PNH clone. Two patients showed more abrupt changes of PNH clone in monocytes than in red blood cells or in granulocytes. However, in the other patient, a significant trend found in only PNH clone of RBC. Conclusion The types of PNH clone observed in each disease group showed different characteristics. PNH clone was identified in 5 of 6 PNH population detected AA cases, whereas minor PNH clones were observed in all 5 PNH population detected cytopenia cases. Four minor PNH clones and two PNH clones were discovered in 6 PNH population detected AML cases. However, all observed PNH clones observed in AML cases were monocyte. Monocyte gating with CD45 and LS not only underestimated the proportion of monocyte in total WBCs but also showed a high false positive rate of 60% in detecting PNH clone. In contrast, the CD14/CD64 gating method can accurately measure the monocyte population and avoid making a false positive measurement of PNH clone. In addition, in monitoring PNH patients, the measurement of the PNH clone in monocyte tends to be more sensitive to change of PNH clone size than those measured in RBC or granulocytes. In conclusion, the gating using CD14 and CD64 is significantly valuable in flow cytometric diagnosis for detecting the PNH clone in diagnosing new patents as well as monitoring of PNH patients. Disclosures No relevant conflicts of interest to declare.
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Kalams, S. A., R. P. Johnson, A. K. Trocha, M. J. Dynan, H. S. Ngo, R. T. D'Aquila, J. T. Kurnick, and B. D. Walker. "Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire." Journal of Experimental Medicine 179, no. 4 (April 1, 1994): 1261–71. http://dx.doi.org/10.1084/jem.179.4.1261.
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Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.
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Taylor, Brian J., Ming Ye, Erin R. Strachan, Tara M. Tiffinger, Andrew R. Belch, and Linda M. Pilarski. "Clonal Analysis of IgM Cells in Multiple Myeloma Patients Suggests the Co-Existence of Normal and Malignant Arms of the Myeloma Clone." Blood 104, no. 11 (November 16, 2004): 1415. http://dx.doi.org/10.1182/blood.v104.11.1415.1415.
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Abstract Analysis of immunoglobulin V genes, which undergo stepwise changes during B cell differentiation such as VDJ rearrangement, somatic hypermutation, and class switch recombination, provides insight into the point of transformation of B cell tumors. In Multiple Myeloma (MM), clonotypic VDJ sequences of malignant plasma cells are mutated, homogeneous, and associated with post-switch constant regions (either IgG or IgA, called the clinical isotype), suggesting the malignant arm of the MM clone arises from transformation events in the late stages of the germinal centre reaction. By contrast, the existence of clonotypic VDJ associated with pre-switch IgM is well established, and we have shown persistent clonotypic IgM is associated with advanced disease at diagnosis and poor survival in MM. Whether clonotypic IgM cells represent a malignant progenitor or a non-malignant population that parallels disease severity is unclear. To address these possibilities, we focused our analysis of clonotypic VDJ mutation profiles on IgM+ cells sorted by immunomagnetic separation from MM patient peripheral blood cells (PBMC). IgM clonotypic transcripts were amplified by hemi-nested RT-PCR targeting the CDR2-C mu constant region in IgM+ cells from 4/7 patients. These products were cloned, and 122, 28, 27, and 25 IgM clonotypic colonies were identified by specific CDR2/CDR3 PCR for patients 1–4 respectively. Each of these clones was sequenced, and mutations were identified by comparison with the closest germline V gene and tumor derived plasma cell VDJ sequences. An average mutation frequency of 0.005, significantly greater than the Taq error rate, was obtained for the 250–280 bp fragment downstream of CDR2, including the D-J-C mu region. Typically, MM clones were observed with 1–2 mutations in this region, many localizing to the D-J-C mu region. Small deletions that preserve reading frame were also observed in the D region of single clones of patients 1 and 4 respectively. The detection of intraclonal heterogeneity amongst clonotypic IgM cells may reflect a normal arm of the myeloma clone that co-exists with the post-switch malignant arm. In previous work examining bulk PBMC populations we had detected diversified clonotypic cells in the non-clinical isotype compartment of one patient, but, in accordance with studies performed by several other groups, were unable to detect diversified pre-switch counterparts. In this work we have focused on IgM+ MM B cells, a compartment of the MM clone that may remain driven by antigenic selection and undergo persistent clonal expansion. Our analysis gives insight into the nature of this proposed normal arm of the myeloma clone, revealing two coexisting subsets of pre-switch clonotypic IgM cells: a major set exhibiting homogeneity, identity with post-switch tumor VDJ, and questionable transformation status, and a minor clonally heterogeneous set which may represent the pre-malignant clone from which myeloma arose.
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Tsujimoto, H., and B. S. Gill. "Repetitive DNA sequences from polyploid Elymus trachycaulus and the diploid progenitor species: detection and genomic affinity of Elymus chromatin added to wheat." Genome 34, no. 5 (October 1, 1991): 782–89. http://dx.doi.org/10.1139/g91-122.
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A set of four repetitive DNA clones, pEt1, pEt2, pCb1, and pCb3, were isolated from SH-genome polyploid Elymus trachycaulus and H-genome diploid Critesion bogdanii. The clone Et1 represents a tandemly arranged telomeric sequence. Et2 represents tandem repeats interspersed along the entire length of individual chromosomes. The Cb1 sequence was more evenly dispersed. The Et1 clone shared homology with a 350 base pair family of rye sequences. The Cb3 sequence was evenly distributed in S- and H-genome species. All the repetitive DNA sequences were excellent markers for the specific detection and genomic affinity of Elymus chromatin added to wheat. All clones showed intragenomic variation in copy number and chromosomal location. Based on the analysis of this variation, we conclude that E. trachycaulus most probably originated from putative diploid H- and S-genome species resembling Critesion californicum and Pseudoroegneria spicata, respectively.Key words: wheatgrass, wheat–Elymus hybrid, addition lines, polyploidy, restriction fragment length polymorphism.
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Gottlob-McHugh, Sylvia G., and Douglas A. Johnson. "Detection of a subfamily of genes within the soybean nodulin-A multigene family." Canadian Journal of Botany 69, no. 12 (December 1, 1991): 2663–69. http://dx.doi.org/10.1139/b91-334.
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A soybean cDNA clone, 15-9-A, corresponding to a new nodule-specific gene was isolated. 15-9-A corresponds to an abundantly transcribed 1.0-kb mRNA that could first be detected in root nodules at about 10 days following inoculation with Bradyrhizobium japonicum. This clone was found to be very closely related to, but distinct from, Ngm-20, a member of a soybean nodulin multigene family. We have used oligonucleotide probes to detect an additional closely related gene, Ngm-20r, which is expressed as a 0.8-kb mRNA. 15-9-A, Ngm-20, and Ngm-20r appear to form a subfamily within the larger nodulin multigene family. Results derived from sequence analysis and hybridization experiments suggest that gene conversion may have played a role in the evolution of this subfamily. Key words: cDNA clone, gene conversion, multigene family, nodule specific, soybean.
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Kriangkum, Jitra, Carina Debes Marun, Sarah Motz, Andrew Belch, and Linda M. Pilarski. "Second Clones In Molecularly-Defined Biclonal Multiple Myeloma Are Derived From Unrelated B Cells, Can Be Present at High Frequency and Exhibit Chromosomal Abnormalities." Blood 116, no. 21 (November 19, 2010): 2981. http://dx.doi.org/10.1182/blood.v116.21.2981.2981.
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Abstract Abstract 2981 Typically MM involves a single monoclonal protein, predicting a single CDR3 peak. We have shown that a sizeable number of patients do not fit this pattern. RT-PCR and genomic PCR (gPCR) were performed using universal primers that bind to FR3/JHc or FR3/CH1 to identify the monoclonal CDR3. In a cohort of 49 MM patients, 10 (20%) exhibited two CDR3 peaks, one of the clinically identified MM clone and another “second” clone. With one exception, a biclonal gammopathy, these second clones were not detected serologically by serum protein electrophoresis or immunofixation. Molecular biclonality is categorized into 3 groups based on the isotype of second clones: group I (1 IgD and 4 IgG MM) where second clone has a pre-switch isotype; group II (3 IgG MM) where the second clone has a post-switch isotype and group III (2 IgA MM) where the second clone exhibits ongoing class switch recombination. All MM clones undergo somatic hypermutation (2.44-15.03%, median = 8.55%). For second clones, 2/10 are unmutated (0-1.05%, below 2% cut off) and 8/10 are mutated (2.44-13.54%, median = 5.90%). The VH3 gene family was utilized in 6/10 of MM clones and 5/10 of second clones. For 10/10 patients, the clinical MM clones are unrelated to second clones as shown by different combinatorial VDJ, different length of CDR3 and different VDJ junction. Biclonal distribution was compared by grading of CDR3 peak heights generated by gPCR. For 9/10 patients, the MM clone is most frequent in BM, 3 of which also predominated in blood. For 6/10 patients, second clones predominated in blood. Q-PCR of mononuclear cells (MNC) showed that second clones ranged from 0.06–24% (median = 1%, n=7) in BM and 0.1–31% (median = 1.45%, n=6) in blood. Chromosomal abnormalities in plasma cells (PC) were seen in 8/8 patients analysed; among these, 4 had t(11;14). Other genetic abnormalities included deletion 13q14, amplification 1q21, trisomy and tetrasomy of chromosome 1. For 4/5 patients where second clones could be distinguished from the MM clone by IgH immuno-FISH, chromosomal abnormalities were detected in the second clone, which in some cases were different from those in the MM clone. Longitudinal analysis was studied in 3 patients. For case 1, a biclonal gammopathy, the IgGλ clone exhibited deletion 13q14 and amplification 1q21, and was dominant in a sternal biopsy (IgGλ/IgGκ = 3.6%/<0.01% MNC; PC<5%MNC); the IgGκ clone exhibited t(11;14) and was the dominant clone in sequential iliac biopsies (IgGλ/IgGκ = 0.3%/3.3% MNC; PC=5-10%MNC). Both clones responded to treatment as measured by Q-PCR, and CDR3 analysis. Case 2 had an IgG MM clone and an IgM second clone; the IgG clone had del13q14 and t(11;14) while the second clone had no detected chromosomal abnormalities. Both clones persisted for at least 2 years during which the MM clone comprised 26–41% of blood MNC while the second clone comprised 12–31% of MNC. The abnormally high abundance suggested a pathological role for the second clone in this patient. Case 3 had an IgD MM clone with an IgM second clone utilizing unmutated VH1-69/DH3-3/JH6 and a long CDR3. Several chromosomal abnormalities including t(11;14), trisomy 11, 14 and 17 were observed in IgD+ and IgM+ cells. The second clone is 0.06% in BM and 1.2% in pre-treatment blood, higher than the expected frequency of antigen-specific B cells. The second clone persisted over 7 months although both MM and second clones responded to treatment. Overall, our studies show that molecular biclonality is frequent in MM, and some second clones have a high frequency in blood and/or BM. Biclonal detection in multiple specimens suggests the persistence of partner clones. The unrelated VDJ origin and distinct chromosomal abnormalities between MM and second clones suggests that they may arise from different transformation events. The particularly high abundance of the second clone in some patients predicts a direct contribution to pathogenesis and disease outcome. Although the clinical significance of second clones remains unclear, the high frequency for some, the presence of chromosomal abnormalities and their persistence during therapy suggest that they may be clinically significant. The prevalence of MM patients having genetically abnormal and frequent second clones suggests that MM may be characterized by more than one transformation event, with clonal dominance defining the clinically identified MM clone. Disclosures: No relevant conflicts of interest to declare.
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Urbano, R., B. Palenik, C. J. Gaston, and K. A. Prather. "Detection and phylogenetic analysis of coastal bioaerosols using culture dependent and independent techniques." Biogeosciences 8, no. 2 (February 10, 2011): 301–9. http://dx.doi.org/10.5194/bg-8-301-2011.
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Abstract. Bioaerosols are emerging as important yet poorly understood players in atmospheric processes. Microorganisms can impact atmospheric chemistry through metabolic reactions and can potentially influence physical processes by participating in ice nucleation and cloud droplet formation. Microbial roles in atmospheric processes are thought to be species-specific and potentially dependent on cell viability. Using a coastal pier monitoring site as a sampling platform, culture-dependent (i.e. agar plates) and culture-independent (i.e. DNA clone libraries from filters) approaches were combined with 18S rRNA and 16S rRNA gene targeting to obtain insight into the local atmospheric microbial composition. From 13 microbial isolates and 42 DNA library clones, a total of 55 sequences were obtained representing four independent sampling events. Sequence analysis revealed that in these coastal samples two fungal phyla, Ascomycota and Basidiomycota, predominate among eukaryotes while Firmicutes and Proteobacteria predominate among bacteria. Furthermore, our culture-dependent study verifies the viability of microbes from all four phyla detected through our culture-independent study. Contrary to our expectations and despite oceanic air mass sources, common marine planktonic bacteria and phytoplankton were not typically found. The abundance of terrestrial and marine sediment-associated microorganisms suggests a potential importance for bioaerosols derived from beaches and/or coastal erosion processes.
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Morgan, J. Alun W., Martin Sergeant, Debbie Ellis, Margaret Ousley, and Paul Jarrett. "Sequence Analysis of Insecticidal Genes fromXenorhabdus nematophilus PMFI296." Applied and Environmental Microbiology 67, no. 5 (May 1, 2001): 2062–69. http://dx.doi.org/10.1128/aem.67.5.2062-2069.2001.
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ABSTRACT Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared inEscherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC50] of 2 to 6 μg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1gene from the bacteriophage lambda P L promoter were shown to have insecticidal activity (LC50 of 112 μg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilusPMFI296.
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Pierobon, Mariaelena, Elisa Baldelli, K. Alex Hodge, Maria Isabella Sereni, Vienna Ludovini, Guido Bellezza, Lucio Crino, Lance A. Liotta, and Emanuel Petricoin. "Development of a quantitative PD-L1 assay using laser capture microdissection (LCM)-based reverse phase protein microarray (RPPA) workflow: Implications for precision medicine." Journal of Clinical Oncology 36, no. 5_suppl (February 10, 2018): 35. http://dx.doi.org/10.1200/jco.2018.36.5_suppl.35.
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35 Background: FDA approved IHC-based companion/complementary assays are routinely used to measure PD-L1 for treatment selection. However, IHC cut-point values vary across platforms and are based on subjective analysis that requires antigen retrieval methods. Even in highly selected populations of PD-L1 “positive” patients, clinical benefits are seen only in subgroups of patients. We tested the feasibility of utilizing LCM and RPPA as a new methodology for quantitative, operator independent measurements of PD-L1 on tumor cells. Methods: PD-L1 quantification by RPPA was compared to IHC on 23 lung cancers (LC). Tumor cells were isolated from the surrounding microenvironment using LCM. The E1L3N clone from Cell Signaling was used to quantify PD-L1 expression by RPPA and IHC. Reproducibility across antibody clones was then assessed on LCM procured tumor epithelia from 10 FFPE LC and 71 snap-frozen ovarian cancers (OC). PD-L1 measurements were compared between the Cell Signaling E1L3N and the Ventana SP-142 clone. A quantitative PD-L1 calibrated assay was developed and assessed with the pilot population data. Results: Of the 23 LC, 5 were PD-L1 positive by IHC. The 5 IHC-positive patients had the greatest level of PD-L1 expression by RPPA indicating that the RPPA platform correlates with IHC. However, RPPA quantification showed a dynamic range ~ 4-fold across IHC negative samples. Correlation between PD-L1 detection with the E1L3N and the SP-142 clone in the OC was significant (R2 = 0.85), indicating great concordance across clones. Results were confirmed in the 10 LC samples (R2 = 0.87). Conclusions: The LCM-RPPA workflow captures PD-L1 expression on a continuous quantitative scale. This quantitative output correlated with IHC although it captures a much broader dynamic range in both IHC negative and positive populations. PD-L1 quantification by LCM-RPPA may be less dependent upon the clone used for the detection than IHC. Because the detection is unconstrained by antigen retrieval issues as well as subjectivity of IHC interpretation, this approach may generate a more accurate cut-point of therapeutic response prediction.